Valor diagnostico de paineis de PCR em tempo-real para detectar a prevalência de agentes etiológicos de diarréia em cães

Orientadora : Profª Drª Simone Tostes de Oliveira StedileDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Agrárias, Programa de Pós-Graduação em Ciências Veterinárias. Defesa: Curitiba, 24/03/2014Inclui referênciasÁrea de concentração : Ciências veterináriasResumo: Diarréia é uma das queixas mais comuns apresentadas aos veterinários diariamente, onde muitas vezes o manejo e diagnóstico definitivo da mesma é um grande desafio, devido a sua variedade de patógenos, ausência de sinais clínicos específicos e presença concomitante de co-infecções por vírus, bactérias e protozoários. Como as doenças infecciosas e parasitárias estão entre as causas mais comuns de diarreia e os testes diagnósticos, assim como os respectivos tratamentos frequentemente variam de acordo com a etiologia da mesma, fazer esta caracterização inicial é importante. Raramente a realização de vários testes diagnósticos são realizados para identificar todas as possíveis causas da diarreia em cães. Isso pode ser em parte porque os métodos tradicionais para a identificação de infecções gastrointestinais são caros, de baixa sensibilidade diagnóstica ou lentos para produzir resultados. A falta de um diagnóstico definitivo, a falha no tratamento com diarréia persistente ou recorrente e a despesa com medicamentos ineficazes, podem levar à insatisfação do cliente, resistência medicamentosa, bem como colocar em risco a saúde do animal. O objetivo deste estudo foi o uso de um painel de PCR em tempo-real e do parasitológico de fezes para pesquisa da prevalência de patógenos e co-infecções em uma população de cães com e sem diarreia atendidos em um hospital veterinário particular. Para tanto, o trabalho foi dividido em dois capítulos, sendo o primeiro um artigo de revisão preparado para a revista Clínica Veterinária, sobre o uso do PCR em tempo-real no diagnóstico de doenças infecciosas em cães e gatos e comparação desta com outras técnicas de diagnóstico. O segundo capítulo compreende a tabulação dos resultados obtidos com as análises realizadas e confecção de um artigo submetido e aceito pelo jornal BMC Veterinary Research. As amostras de fezes foram testadas para o vírus da cinomose canina, coronavírus canino, parvovírus canino tipo 2 (CPV-2), Clostridium perfringens alfa toxina (CPA), Cryptosporidium spp., Giardia spp. e Salmonella spp. através de técnicas moleculares e a pesquisa de larvas, ovos, cistos e trofozoítos por meio do parasitológico de fezes. Do total das 104 amostras de fezes diarreicas e 43 controles, 71/104 (68.3%) dos cães com diarreia foram positivos para pelo menos um patógeno, onde destes, infecção única foi observada em 39/71 (54,9%) e coinfecção em 32/71 (45,1%), incluindo 21/32 (65,6%) dos cães com duas, 5/32 (15,6%) com três e 6/32 (18,8%) com quatro infecções. No grupo controle, 13/43 (30,2%) dos cães foram positivos, todos com infecção única. Os patógenos mais prevalentes nos cães com diarreia foram CPA (40/104 cães, 38.5%), CPV-2 (36/104 cães, 34.6%), e Giardia spp. (14/104 Cães, 13.5%). O CPV-2 foi o patógeno mais prevalentemente associado a co-infecções, principalmente com os agentes CPA, Cryptosporidium spp. e Giardia spp. Palavras-chave: Cães, Co-infecção, Prevalência, Diarreia, Painel, PCR em temporealAbstract: Diarrhea is one of the most common reasons pets are taken to the veterinary clinician daily. Definitive diagnosis and management often pose great challenges due to its variety of pathogens, absence of specific clinical signs and concomitant presence of co-infections with viruses, bacteria and protozoa. Since infectious and parasitic diseases are among the most common causes of diarrhea and diagnostic tests, as well as their treatments, often vary according to the etiology of the disease, prompt characterization is important. Rarely various diagnostic tests are performed to identify all possible causes of diarrhea in dogs. This may be partly because traditional methods for identification of gastrointestinal infections are expensive, have low diagnostic sensitivity and are time-consuming. The lack of a definitive diagnosis, failing to treat persistent or recurrent diarrhea and spending on ineffective drugs, can lead to customer dissatisfaction, drug resistance, as well as put the animal's health at risk. The aim of this study was the use of a Real-Time PCR panel and parasitological stool examination to survey prevalence of pathogens and co-infections in a population of dogs with and without diarrhea seen at a private veterinary hospital, as well as frequency in different countries. Therefore, the work was divided in two chapters, the first a review article prepared for the Clínica Veterinária journal on the use of Real-Time PCR for diagnosis of infectious diseases in dogs and cats and comparison with other diagnostic techniques. The second chapter contains a tabulation of the results obtained from the analyses, and the preparation of a paper submitted and accepted by the BMC Veterinary Research journal. Stool samples were tested for canine distemper virus, canine coronavirus, canine parvovirus type 2 (CPV-2), Clostridium perfringens alpha toxin (CPA), Cryptosporidium spp., Giardia spp. and Salmonella spp. through molecular techniques and examined for larvae, eggs, cysts and trophozoites by parasitological stool examination. Of the total of 104 samples of diarrheal stools and 43 controls, 71/104 (68.3 %) of the dogs with diarrhea were positive for at least one pathogen. For positive samples, single infection was observed in 39/71 (54.9%) and co-infection in 32/ 71 (45.1 %), including 21/32 (65.6%) dogs with two, 5/32 (15.6%) dogs with three and 6/32 (18.8%) dogs with four infections. In the control group, 13/43 (30.2 %) dogs were positive, all with single infections. The most prevalent pathogens observed in dogs with diarrhea were CPA (40/104 dogs, 38.5 %), CPV-2 (36/104 dogs, 34.6 %) and Giardia spp. (14/104 dogs, 13.5 %). CPV - 2 was the pathogen most prevalently associated with co- infections, especially associatd with CPA, Cryptosporidium spp. and Giardia spp. Keywords: Dogs, Co-infection, Prevalence, Diarrhea, Panel, Real-Time PCR

Presence of infectious agents and co-infections in diarrheic dogs determined with a real-time polymerase chain reaction-based panel

BACKGROUND: Infectious diarrhea can be caused by bacteria, viruses, or protozoan organisms, or a combination of these. The identification of co-infections in dogs is important to determine the prognosis and to plan strategies for their treatment and prophylaxis. Although many pathogens have been individually detected with real-time polymerase chain reaction (PCR), a comprehensive panel of agents that cause diarrhea in privately owned dogs has not yet been established. The objective of this study was to use a real-time PCR diarrhea panel to survey the frequencies of pathogens and co-infections in owned dogs attended in a veterinary hospital with and without diarrhea, as well the frequency in different countries. Feces samples were tested for canine distemper virus, canine coronavirus, canine parvovirus type 2 (CPV-2), Clostridium perfringens alpha toxin (CPA), Cryptosporidium spp., Giardia spp., and Salmonella spp. using molecular techniques. RESULTS: In total, 104 diarrheic and 43 control dogs that were presented consecutively at a major private veterinary hospital were included in the study. Overall, 71/104 (68.3%) dogs with diarrhea were positive for at least one pathogen: a single infection in 39/71 dogs (54.9%) and co-infections in 32/71 dogs (45.1%), including 21/32 dogs (65.6%) with dual, 5/32 (15.6%) with triple, and 6/32 (18.8%) with quadruple infections. In the control group, 13/43 (30.2%) dogs were positive, all with single infections only. The most prevalent pathogens in the diarrheic dogs were CPA (40/104 dogs, 38.5%), CPV-2 (36/104 dogs, 34.6%), and Giardia spp. (14/104 dogs, 13.5%). CPV-2 was the most prevalent pathogen in the dual co-infections, associated with CPA, Cryptosporidium spp., or Giardia spp. No statistical difference (P = 0.8374) was observed in the duration of diarrhea or the number of deaths (P = 0.5722) in the presence or absence of single or co-infections. CONCLUSIONS: Diarrheic dogs showed a higher prevalence of pathogen infections than the controls. Whereas the healthy dogs had only single infections, about half the diarrheic dogs had co-infections. Therefore, multiple pathogens should be investigated in dogs presenting with diarrhea. The effects of multiple pathogens on the disease outcomes remain unclear because the rate of death and the duration of diarrhea did not seem to be affected by these factors

Detecção precoce e quantificação de vírus da cinomose por PCR quantitativa em Tempo Real (qPCR) em diferentes tecidos e fluidos de um cão

A cinomose é uma doença de desafio diagnóstico, especialmente quando não há histórico de vacinação. O objetivo deste estudo foi detectar e quantificar partículas virais de cinomose em diferentes fluidos e tecidos biológicos de um cão, determinando o melhor tecido para diagnóstico viral ante mortem na fase de viremia. Atendeu-se um cão adulto com manifestações clínicas inespecíficas e corpúsculos de Sinegaglia Lentz em linfócitos. Amostras post mortem foram submetidas a PCR em tempo real (qPCR), que demonstrou RNA viral em concentrações de (x105) em líquor (1.216), bexiga (1.009), cérebro (605), sangue (572), cerebelo (523), rins (373), fígado (257), pulmões (191), estômago (154), terceira pálpebra (70) e urina (2,1). A técnica de qPCR permitiu confirmar a infecção pelo vírus, descartando vacinação recente. A amostra de líquor mostrou-se representativa para diagnóstico molecular de fase aguda de cinomose no animal estudado

Safety of systemic anti-cancer treatment in oncology patients with non-severe COVID-19: a cohort study.

BACKGROUND: The viral pandemic coronavirus disease 2019 (COVID-19) has disrupted cancer patient management around the world. Most reported data relate to incidence, risk factors, and outcome of severe COVID-19. The safety of systemic anti-cancer therapy in oncology patients with non-severe COVID-19 is an important matter in daily practice. METHODS: ONCOSARS-1 was a single-center, academic observational study. Adult patients with solid tumors treated in the oncology day unit with systemic anti-cancer therapy during the initial phase of the COVID-19 pandemic in Belgium were prospectively included. All patients (n = 363) underwent severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) serological testing after the first peak of the pandemic in Belgium. Additionally, 141 of these patients also had a SARS-CoV-2 RT-PCR test during the pandemic. The main objective was to retrospectively determine the safety of systemic cancer treatment, measured by the rate of adverse events according to the Common Terminology Criteria for Adverse Events, in SARS-CoV-2-positive patients compared with SARS-CoV-2-negative patients. RESULTS: Twenty-two (6%) of the 363 eligible patients were positive for SARS-CoV-2 by RT-PCR and/or serology. Of these, three required transient oxygen supplementation, but none required admission to the intensive care unit. Hematotoxicity was the only adverse event more frequently observed in SARS-CoV-2 -positive patients than in SARS-CoV-2-negative patients: 73% vs 35% (P < 0.001). This association remained significant (odds ratio (OR) 4.1, P = 0.009) even after adjusting for performance status and type of systemic treatment. Hematological adverse events led to more treatment delays for the SARS-CoV-2-positive group: 55% vs 20% (P < 0.001). Median duration of treatment interruption was similar between the two groups: 14 and 11 days, respectively. Febrile neutropenia, infections unrelated to COVID-19, and bleeding events occurred at a low rate in the SARS-CoV-2-positive patients. CONCLUSION: Systemic anti-cancer therapy appeared safe in ambulatory oncology patients treated during the COVID-19 pandemic. There were, however, more treatment delays in the SARS-CoV-2-positive population, mainly due to a higher rate of hematological adverse events